Compounds for PDT

ABSTRACT

A tetrakis(hydroxyphenyl)chlorin, bacteriochlorin or isobacteriochlorin, derivatised at one or more of the hydroxy groups by addition reaction with a diisocyanate, diisothiocyanate or isocyanate-isothiocyanate at one isocyanate or isothiocyanate group thereof, the other isocyanate or isothiocyanate group being itself derivatised by addition reaction with the hydroxy group of an w-alkylated or acylated poly(alkylene oxide) or to a hydroxy group of a link residue itself carrying a residue of such poly(alkylene oxide).

This application is the US national phase of international applicationPCT/GB01/01010 filed 8 Mar. 2001, which designated the US.

FIELD OF INVENTION

The invention relates to poly(alkylene oxide) substitutedphotosensitising compounds and to their use in photodynamic therapy ofcancerous and other diseased tissues.

BACKGROUND

Photodynamic therapy (PDT) involves the administration of aphotosensitising agent for localisation in target diseased tissuefollowed by irradiation of the target tissue containing the compoundwith light of a specific and appropriate wavelength. The resultingphotoactivated compound, in the presence of oxygen, leads to necrosis ofthe tissue.

The success of this modality is dependent on administration of acompound that is selectively retained in tumour tissue as compared tonormal tissue. Thus, on irradiation of the tumour with light of thephotoactivating wavelength, the amount of damage caused by necrosis isproportionately higher than that in normal tissue. However, some normaltissue damage typically occurs and one specific side effect seen withthe use of many photosensitisers is redness and swelling of the skin onsubsequent exposure to normal lighting levels and particularly sunlight.Such side effects are minimised by keeping the patients in subdued lightfor a prolonged period after treatment, consequently restricting theirquality of life. A more efficient delivery of the photosensitiser intotumour tissue, thus providing a much higher tumour to normal tissueratio of drug concentration could dramatically reduce the potential forskin side effects with this treatment.

A group of photosensitising agents have previously been the subject ofpatents EP 0 337 601 and U.S. Pat. No. 4,992,257. These compounds aredihydroporphyrins (chlorins) (1) and the correspondingtetrahydroporphyrins (bacteriochlorins) (2) and (3) of the formulae:

wherein each n=1 to 3 and each substituent, R, the same or different, isa hydroxyl (—OH) group, each itself free or substituted with an alkyl oracyl group. Salts, internal salts, metal complexes or hydrates or othersolvates of the compounds are also covered.

The above formulae, it will be appreciated, represent particulartautomers among various possibilities including chlorins as shown below(represented without meso Phenyl groups):

The invention covers all tautomers of the above compounds and is notlimited to those shown in diagrams.

Published Proposals

Modification of compounds by PEGylation, that is the direct or indirectattachment of polyethylene glycol chains (PEG), and in principle otherpoly(alkylene oxide) chains, is known to introduce useful properties.PEG is non-toxic, imparts good water solubility to drug molecules andalters the biodistribution, which can result in a favourablepharmacokinetic profile. The general topic of polyether substitutedanti-tumour agents is described in DKFZ's specification PCT EP 91/00992(WO 91/18630). No particular attention is given to the selection of thelinkage between the polyether chain and the anti-tumour agent, the onlyexample disclosed being a triazine introduced by initial activation ofthe polyether with cyanuric chloride. More recently, DKFZ have describeda method for the production of chlorins and bacteriochlorins containinga polyether (WO 98/01156). The method involves initial attachment of thepolyether to the porphyrin with subsequent reduction to thecorresponding chlorins and bacteriochlorins. Again, no particularattention is given to the nature of the linkage between the polyetherand the anti-tumour agent, the only example disclosed being an amidelink.

PEGylation of compounds (1), (2) and (3) via triazine, ether and esterlinkages has been previously reported by us in PCT GB 95/00998 (WO95/29915). However, lability of the ester linkages and significantdifficulties in the scale up of the triazine and ether linked moietiesseverely limits the practical utility of these compounds.

Enzon have also reported polyether compositions containing isocyanateand/or isothiocyanate groups for covalent attachment to bioeffectingsubstances such as peptides or chemotherapeutics (WO 94/04193). However,in relation to isocyanates and isothiocyanates, coverage is directed tocompounds in which bioeffecting substances are attached to both ends ofthe polyether chain.

Outside the PDT field, hexane-1,6-diisocyanate has been used to link PEGto atropine (Zalipsky et al, Eur. Polym. J. 1983, 19(12), 1177-1183) andto 5-fluorouracil (Ouchi et al, Drug Design and Discovery 1992, 9,93-105). Bayer (U.S. Pat. No. 4,684,728) have reported a process forimproving the solubility in water of a sparingly soluble biologicallyactive compound by reaction to form a derivative carrying the activemoiety, a linking group such as an optionally substituted diisocyanategroup and a polyether chain. No mention is made of any benefit to thetherapeutic profile of such compounds other than the ease of formulationand administration of a water soluble compound.

Discussion of Present Work

Advances in photodynamic therapy for clinical disease treatment,particularly cancer, depend on developing improved photosensitisers. Thedesired characteristics of an ideal photosensitiser include selectivediseased tissue localisation, activation at long wavelengths so thatmaximum depth of tissue penetration is shown, and high efficiency assensitisers. From a formulation and administration point of view, watersolubility is also a beneficial attribute.

Photodynamic therapy is a dual therapy, which consists of the combinedaction of photosensitiser and light. In clinical practice the drug isfirst administered, and then activated by light some time later. Thetime period between administering the drug and applying the light iscalled the drug-light interval. It is desirable to apply light at a timewhen the photosensitiser has accumulated maximally in the target tissueand has been eliminated from the normal surrounding tissue. Theprincipal factor determining the drug-light interval is the drugpharmacokinetic profile which itself varies between every tissue. Thedrug-light interval has to be suitable for clinical practice. From aclinical standpoint, pharmacokinetics which give a maximum drugconcentration in a tumour as soon as possible, for example from a fewhours to at most 3 days, together with rapid elimination from the bodythereafter, would be ideal. This would allow flexibility in schedulingtreatment.

In European Patent Specification 0 337 601 (U.S. Pat. No. 4,992,257),the applicants disclose compounds with many of the desiredcharacteristics, particularly an extremely high photo efficiency, thatis to say the ability to generate free radical species such as singletoxygen through the absorption of light. The long absorption wavelengthsof the molecules, e.g. at 652 nm and 734 nm, penetrates tissueefficiently and thus the sensitisers can be used to treat deep tumours.

However, the disclosed compounds do not fulfil all the requirementsequally well. A residual disadvantage is the degree of normal tissuephotosensitivity, particularly of the skin, that occurs followingadministration of the sensitiser. This arises from unwanted depositionof the sensitiser in the skin and other normal tissue and is aconsequence of imperfect tumour targeting by the drug. The skinphotosensitivity can last up to 4 weeks depending on the drug doseadministered. At the usual clinical dose of 0.15 mg kg⁻¹ skinsensitivity of, for example, m-THPC (a tetraphenyl chlorin derivative inwhich each phenyl group carries a m-hydroxy group) lasts for 2-3 weeks.This limits the patient's freedom and is an undesirable restriction.

We have sought ways of overcoming normal tissue photosensitivity,particularly of the skin, by converting m-THPC to a polyethylene glycolderivative. This ‘PEGylation’ profoundly alters the bodily distributionin a favourable way by increasing tumour targeting, and at the same timereducing uptake to the skin. The distribution of the compound is alteredby PEGylation, due to hydrogen bonding of water to oxygen on thepolyethylene glycol chains when the compound is injected into the blood.A ‘water envelope’ forms around the photosensitiser and prevents thecompound sticking to the endothelium of blood vessel walls and in turnpassing into the surrounding tissue including the skin. This favoursuptake to the tumour through the enhanced permeability and retention(EPR) effect. Tumours have a disturbed vasculature and lymphaticdrainage, leading to increased accumulation of substances such as drugsin the tumour compared to normal tissue (R. Duncan and F. Spreafico,Clin. Pharmacokinet. 1994, 27, 290-306). This effect can be enhancedwith higher molecular weight compounds. The net effect of

PEGylation is that the compound is favourably redirected from the skinand other normal tissues towards the tumour, thus reducing the degree ofskin sensitivity.

It has been confirmed experimentally, for example, that PEGylation canproduce a favourable tissue re-distribution. This was shown in a mouseexperimental model in which an outstanding difference between muscle andtumour photosensitivity during PDT was found for the triazine-linkedderivative (Grahn et al., Proc. SPE 1997, 3191, 180-6). Three days afterthe PEGylated m-THPC was administered it was found that the muscle wasno longer photosensitive, while the tumour retained its maximumsensitivity to light for at least 15 days after drug administration.This presented ample time for tumour-selective treatment, but didindicate the less desirable characteristic of tumour persistence withthis derivative.

Other work previously performed with triazine-linked PEG derivatives[applicants PCT Patent specification WO 95/29915, (PCT/GB95/00998)]confirmed that the pharmacokinetics with the triazine linkage wererather too prolonged for routine clinical use. In particular, excretionfrom the liver was very slow indeed, which is undesirablepharmaceutically.

An alternative linker to the triazine molecule was sought, including aglycidic ether with amino PEG and also an hexylbiscarbamate linker. ThePEGylated derivatives of m-THPC with triazine and carbamate links havevery different and unexpected pharmacokinetics to each other and m-THPC.The triazine-linked compound (SPC 0038B) is excreted from the liver moreslowly than m-THPC, while the carbamate derivative (SPC 0172) isexcreted in a comparable period to m-THPC. The solubilities of thecompounds, and hence ease of pharmaceutical preparation, were enhancedto levels of up to 52 mg/mL in water, compared to m-THPC, which isinsoluble in aqueous solvents.

The linker group should provide a stable point of PEG attachment,permitting reasonable in vivo circulation and should be available via apractical and robust synthesis. It should not, however, affect the PDTefficiency of the drug molecule. The method of Zalipsky was modified andutilised for a two step synthesis from the chlorin and bacteriochlorinmolecules to their PEGylated derivatives. Analysis of these compoundsusing gel permeation chromatography (GPC) allowed separation of lesserPEGylated forms, but high performance liquid chromatography (HPLC)proved superior with separation between the peaks of 0.8 min. Reactionproducts were also analysed by UV/Visible spectroscopy, which gave aquantitative measurement of molecular weight (mw) using the formula:Apparent mw=mw (chlorin)×A _((1%, 1 cm)) (chlorin)/A _((1%, 1 cm)) (PEGchlorin).

The applicants work has thus built on previous work in trying to developan ideal sensitiser. Unpredictably, the carbamate-linked polyethyleneglycol derivatives have an excellent and preferred pharmacokineticprofile from a clinical point of view and exhibit less potential tocause cutaneous photosensitivity. Furthermore, studies in Balb/c micebearing colo26, a murine colorectal cancer, show that the photodynamiceffect of the carbamate-linked derivative in tumour is maximum at 2 daysand that it has the same PDT activity as m-THPC itself This isconsiderably more active than the triazine-linked compound. Thusoverall, the PEGylated carbamate-linked compound appears to add newfeatures, which enhance the desired characteristics of the sensitiser.

The Invention

The present invention summarised below and set out in the claims thusconcerns the derivatisation or partial derivatisation of the phenolicgroups of compounds of formulae (1), (2) and (3) with poly(alkyleneoxides) using a carbamate or thiocarbamate link:

(X═O, S) formed by addition reaction of the compounds with an isocyanate(—N═C═O) or isothiocyanate (—N═C═S) group of a diisocyanate,diisothiocyanate or an isocyanate-isothiocyanate, the poly(alkyleneoxide) chain being attached directly or indirectly by addition at theother group and its terminal hydroxyl group being etherified oresterified with for example a C₁₋₁₂ alkyl or acyl group of which methylis the most preferred.

The reactions, which may be carried out in any convenient order, resultin compounds of formulae:

and imino-tautomers thereof wherein n=1 to 3 and R′ may be the same ordifferent, is a hydroxyl (—OH) group, each itself free or substitutedwith an alkyl or acyl group, but in at least one, preferably more thanone instance is as follows:

where:

-   -   (i) each X, the same or different, is O, S;    -   (ii) Y is O (carbamate or thiocarbamate link);    -   (iii) A is a hydrocarbon group containing 2 to 40 carbon atoms,        preferably 4 to 20 carbon atoms and very preferably 6 carbon        atoms. This group may be branched or unbranched, cyclic or        acyclic, saturated or unsaturated, aliphatic or aromatic;    -   (iv) B is an optional group ((CH₂)_(p)—O)_(q) where p=1 to 4;        q=0,1;    -   (v) D is a poly(alkylene oxide), preferably polyethylene glycol,        with an average molecular weight of at least 200 and not more        than 40,000, preferably 750 to 20,000 and very preferably 2,000        to 5,000 Da;    -   (vi) E is an alkyl or acyl group containing 1 to 12 carbon        atoms, preferably a methyl group.

In any of the above compounds derivatives such as salts with mineralacids (e.g. hydrochlorides, sulphates), internal salts, metal complexes(e.g. with Zn, Ga), or hydrates and other solvates may be formed.

Suitable diisocyanates include butane-1,4-diisocyanate,hexane-1,6-diisocyanate, octane-1,8-diisocyanate, dodecane-1,12-iisocyanate, 2-methylpentane-1,5-diisocyanate,toluene-2,4-diisocyanate, toluene-2,6-diisocyanate,cyclohexane-trans-1,4diisocyanate,dicyclohexylmethane-4,4′-diisocyanate,diphenylmethane-3,4′-diisocyanate, xylene diisocyanate and2,4,4-trimethylhexylmethylene diisocyanate. Correspondingdiisothiocyanates and isocyanate-isothiocyanates are also appropriate.The most preferred linker is hexane-1,6-diisocyanate.

Chemistry

Compounds of types (12), (13) and (14) may be prepared in a two stepprocess.

-   (i) activation of poly(alkylene oxide) by reaction with a    diisocyanate, diisothiocyanate or an isocyanate-isothiocyanate (e.g.    hexane-1,6-diisocyanate) in a suitable inert, anhydrous solvent    (e.g. toluene) with or without a catalyst (e.g. dibutyl tin    dilaurate), with or without a tertiary organic base (e.g.    triethylamine) at a temperature between 0 and 110° C.-   (ii) coupling of the activated poly(alkylene oxide) to the reduced    porphyrin in a suitable inert solvent (e.g. toluene) with or without    a catalyst (e.g. dibutyl tin dilaurate), with or without a tertiary    organic base (e.g. triethylamine) at a temperature between 0 and    110° C.

Synthesis of compounds may also be achieved by reversing the order ofthe steps, namely activation of the reduced porphyrin by reaction withthe diisocyanate, diisothiocyanate or isocyanate-isothiocyanate followedby coupling with the poly(alkylene oxide). However, the former approachis preferred.

Routes of Administrations

By parenteral or any other suitable route in per se known manner.

Pharmaceutical Presentations

Any suitable presentation as known in the field, including, but notlimited to:

-   -   i) injectable solution    -   ii) freeze dried powder for reconstitution and injection    -   iii) infusion solution for addition to saline or other vehicle    -   iv) tablet or capsule for oral administration.

PREPARATIVE EXAMPLES Example 1

7,8-Dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatisedwith ω-methoxy polyethylene glycol (average MW=2000) via biscarbamatelinkages derived from hexane-1,6-diisocyanate.

[Compound (12); n=1: meta substitution on all aryl groups; X═O;A=(CH₂)₆; Y═O; q=0; D=PEG (average MW=2000); E=CH₃]

Part 1: Preparation of Activated mPEG (ω-methoxy Polyethylene Glycol)

A solution of mPEG (average MW=2000, 40 g) in toluene was driedazeotropically for 4 h and added dropwise over a 2 h period to a mixtureof anhydrous toluene (100 mL), hexane-1,6-diisocyanate (16.2 mL) anddibutyl tin dilaurate (0.5 mL). After standing overnight under anhydrousconditions, the product was precipitated by the addition of hexane (200mL). The solid was collected by filtration, reprecipitated fromtoluene/hexane and dried under vacuum. This yielded the product as awhite powder (38 g). Analysis by non-aqueous titration gave anisocyanate assay of 95% of theory. Molecular weight as determined bytitration of NCO groups: mPEG₂₀₀₀-hexylcarbamateisocyanate 2160(requires 2168). IR (nujol, cm⁻¹) 3300 (NH); 2250 (NCO); 1715, 1535(HN—COO); 1110 (CH₂OCH₂); Aδ_(H) (CCl₄) 1.3-1.6 (m, CH₂), 3.6 (s, OCH₂).This material was used immediately in the second part of the synthesis.

Part 2: Coupling of Activated mPEG to m-THPC

A mixture of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin(300 mg) and activated mPEG (as prepared in Part 1) (8.95 g) inanhydrous toluene was stirred overnight under nitrogen at 30-60° C. HPLCanalysis on an aliquot indicated >95% tetraPEGylation. The product wasprecipitated by the addition of hexane to the stirred contents at roomtemperature. The solid was collected by filtration, washed with hexaneand dried under vacuum The product was then purified by reverse phasechromatography, eluting with methanol/water. After removal of themethanol under reduced pressure, the solution was freeze dried to yieldthe product as a dark brown solid.

Example 2

7,8,17,18-Tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrinderivatised with ω-methoxy poly(ethylene glycol) (average MW=2000) viabis carbamate linkages derived from hexane-1,6-diisocyanate.

[Compound (13); n=1; meta substitution on all aryl groups; X═O;A=(CH₂)₆; Y═O; q=0; D=PEG (average MW=2000); E=CH₃]

By replacing 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrinwith 7,8,17,18-tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrinand toluene with 1,4-dioxan as solvent in Example 1, Part 2, the titlecompound was prepared as a brown powder.

Example 3

7,8-Dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin derivatisedwith ω-methoxy poly(ethylene glycol) (average MW=5000) via bis carbamatelinkages derived from hexane-1,6-diisocyanate.

[Compound (12); n=1; meta substitution on all aryl groups; X═O;A=(CH₂)₆; Y═O; q=0; D=PEG (average MW=5000); E=CH₃]

By replacing mPEG (average MW=2000) with mPEG (average MW=5000) inExample 1, Part 1, [Molecular weight as determined by titration of NCOgroups: mPEG₅₀₀₀-hexylcarbamateisocyanate 4944 (requires 5168)] thetitle compound was prepared as a brown powder.

Example 4

7,8,17,18-Tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrinderivatised with ω-methoxy poly(ethylene glycol) (average MW=5000) viabis carbamate linkages derived from hexane-1,6diisocyanate.

[Compound (13); n=1; meta substitution on all aryl groups; X═O;A=(CH₂)₆; Y═O; q=0; D=PEG (average MW=5000); E=CH₃]

By replacing mPEG (average MW=2000) with mPEG (average MW=5000) inExample 1, Part 1 and 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin with7,8,17,18-tetrahydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin inExample 1, Part 2, the title compound was prepared as a brown powder.

Biology

In the following account of work done:

SPC 0172 is carbamate-linked PEG₂₀₀₀ m-THPC, compound name tetrakis(6′-(methoxy PEG 2000 carbamate)1′-isocyanate hexamethylene) derivativeof 7,8dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin. Thiscompound is shown in example 1.

SPC 0038B is a corresponding triazine compound, tetrakis(ω-methoxypolyethylene glycol [MW=2000] triazine of7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin.

m-THPC is temoporfin or7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin, the basis ofSPC 0172 and SPC 0038B.

Spectroscopic Properties of SPC 0172

SPC 0172 has a very similar absorption spectrum to that of SPC 0038Bwhich in turn was similar to that of m-THPC (absorption peaks for m-THPCin the 500-700 nm range at 516, 542, 594 and 650 nm). FIG. 1(Absorbance, A against wavelength, λ) shows the UV-visible absorbancespectra of SPC 0172 and SPC 0038B in 0.25 μM aqueous solution measuredusing a Hitachi U3000 spectrophotometer. SPC 0172 has a somewhat lowermolar extinction coefficient at the red peak than does m-THPC (ca 22,000L mol⁻¹ cm⁻¹ compared to ca 30,000 L mol⁻¹ cm⁻¹).

The fluorescence emission spectrum in ethanol of SPC 0172 is verysimilar to those of SPC 0038B and m-THPC in the same solvent. All threecompounds show a fluorescence emission peak between 655-660 nm whenexcited by light in the Soret band region (405 nm). m-THPC fluorescenceis severely quenched in aqueous solution owing to the formation ofaggregates. Both SPC 0038B and SPC 0172 fluoresce more weakly in aqueoussolution than in ethanol (47% and 36% respectively) indicating that someaggregation or other conformational change takes place when they aredissolved in water. Both the PEGylated photosensitisers exhibit greatlyimproved solubility in water (at least 50 mg/ml) compared to m-THPC,which is insoluble.

Uptake by Tumour Cells in Culture

Colo26 mouse colorectal tumour cells were grown in culture usingstandard methods. Confluent monolayers of the cells kept at 37° C. inthe dark were exposed to 1.5 μM m-THPC, SPC 0038B or SPC 0172 added tothe incubation medium for periods of between 3 and 72 hours. At the endof the set time period, the culture medium containing the addedphotosensitiser was removed and the cells washed with cold phosphatebuffered saline. The cells were freed from the culture flask bytreatment with trypsin solution. The viable cell count was determinedusing a standard haemocytometer and the photosensitiser extracted fromthe cells by treatment with methanol:DMSO (4:1, v:v). Cell extracts werefrozen in liquid nitrogen and stored at −70° C. for later analysis. Thephotosensitiser content of the cell extract was determined by directfluorescence using standard curves of photosensitiser and correcting forextraction efficiency from the cell suspension.

The uptake of the three photosensitisers is shown in FIG. 2 temoporfinuptake by tumour cells in culture, FIG. 3 SPC0172 uptake by tumour cellsin culture and FIG. 4 SPC0038B uptake by tumour cells in culture, all at37° C. (millions of molecules per cell, m against time t (hours)). Theamount of photosensitiser is expressed as the number of molecules ofphotosensitiser (in millions) present within a single cell at each timepoint The data in FIGS. 2 to 4 represents the mean value obtained fromtwo experiments and the bars represent the range. The uptake of m-THPCis rapid, reaching a peak around 24 hours after which thephotosensitiser content of the cells declines. The uptake of SPC 0038B,in contrast is very slow, with a barely detectable increase in cell drugconcentration at 72 hours compared with 3 hours of incubation. After 72hours the cell photosensitiser content is about a factor of 100 lessthan that of m-THPC. The uptake of SPC 0172 shows the same slower uptakeas does SPC 0038B, uptake being relatively linear up to the finalmeasurement point at 72 hours of incubation. However, the amount ofphotosensitiser taken up is greater. After 72 hours of incubation thereis about 5 times as much SPC 0172 in each cell as SPC 0038B, but againthis is much less than the peak cellular m-THPC content.

The mechanism of photosensitiser uptake by cells remains a subject ofstudy. In the case of small hydrophobic sensitisers such as m-THPC,which are generally presented to cells bound to protein or lipoproteinin biological systems, a role for specific lipoprotein receptors hasbeen implicated. The uptake mechanisms of photosensitiser PEG conjugateshave received much less attention, although fluorescence microscopystudies show that the initial intracellular distribution is limited tofoci in the cytoplasm in the vicinity of the plasma membrane suggestingthat endocytosis and sequestration in vesicles plays a part. One way inwhich uptake mechanisms may be studied is by determining the influenceof temperature. In general, active (energy-dependent) uptake processesare inhibited in cells maintained at 4° C. compared to those at 37° C.Passive (energy-independent) uptake processes are much lesstemperature-dependent. The effect of temperature on uptake of the threephotosensitisers at 1.5 μM is shown in Table 1.

TABLE 1 Effect of temperature on photosensitiser uptake by Colo26 cells6 hour uptake 24 hour uptake Rate of uptake 6-24 h (millions ofmolecules (millions of molecules (thousands of molecules per cell) percell) per cell per hour) Compound 4° C. 37° C. 4° C. 37° C. 4° C. 37° C.m-THPC 0.82 79.2 1.33 208 28 7184 SPC 0038B 0.56 0.82 0.64 0.86 4 2 SPC0172 1.08 1.12 1.78 2.08 39 53

It can be seen that at 4° C. the uptake of m-THPC is inhibited by afactor of over 250. Such strong temperature dependence suggests that theuptake of this compound occurs by an active, energy-dependent process.The uptake of SPC 0038B appears to be twice as great in the cold than at37° C. whilst that of SPC 0172 was reduced on cooling to about 75% ofthat at 37° C. The uptake of both PEGylated photosensitisers thereforewas relatively unaffected by temperature compared to the effect withm-THPC. This suggests that SPC 0172 and SPC 0038B are taken up intotumour cells by means of an energy-independent mechanism. SPC 0172 istaken up more efficiently by tumour cells through this mechanism thanSPC 0038B.

Pharmacokinetic Characteristics

An assessment of the relative pharmacokinetic characteristics has beenmade by comparison of the plasma concentration-time profiles for SPC0172, SPC 0038B and m-THPC in mice bearing a subcutaneously implantedtumour.

Adult female Balb/c mice bearing the syngeneic colo26 tumour implantedsubcutaneously were produced using methods previously described (Ansellet al. Lasers in Medical Science 1997, 12, 336-41). Groups of mice ofweight 19-22 g were given the photosensitisers at a dose of 0.88 μmolkg⁻¹ (equivalent to 0.6 mg kg⁻¹ in the case of m-THPC) by injection intothe tail vein. The solutions for injection of each compound wereprepared at a concentration of 0.352 μmol ml⁻¹ so that a typical 20 gmouse would be injected with a volume of 50 μl. For M-THPC, which isinsoluble in water, the injection solution was prepared using a PEG400:ethanol:water vehicle (30:20:50, w/w), whilst SPC 0172 and SPC 0038Bwere prepared in water for injection.

Animals were sacrificed at 6, 24, 72 and 192 hours after injection ofeach photosensitiser. Immediately after sacrifice, blood was obtained bycardiac puncture, and centrifuged at 13,000×g for three minutes. Theresulting supernatant (blood plasma) was aspirated and stored at −70° C.for subsequent analysis. The photosensitiser content of each sample wasmeasured from the fluorescence of an extract obtained usingmethanol:DMSO (3:5, v:v). A 50 μl portion of each extract wastransferred to a disposable fluorescence cuvette and the fluorescencedetermined using an excitation wavelength of 418 nm and emissionwavelength of 650 nm with an eight second response setting. The assaywas calibrated using stock solutions of each compound in methanol,further diluted in methanol:water (1:1, v/v) and extracted withmethanol:DMSO as described above. For each photosensitiser thefluorescence yield was found to be linear up to a sample concentrationof 200 nmoles per ml. The measurement protocol was designed to diluteeach sample to a sufficient extent to avoid interference by endogenouschromophores (absorbance of samples <<0.1 at the excitation wavelength).The results obtained are presented in FIG. 5 (nano-moles per ml, nagainst time, t (hours)) plasma photosensitiser concentrations followinginjection of 0.88 nanomoles of each photosensitiser per g live weight.

The data show that higher plasma levels of SPC 0038B occur in these micecompared to either m-THPC or SPC 0172 given at equimolar dosesintravenously. It is also clear that the plasma concentrations of bothm-THPC and SPC 0172 diminish to background levels more rapidly than SPC0038B. In effect SPC 0038B persists longer and exhibits a longerterminal elimination phase from plasma compared to either SPC 0172 orm-THPC. The data indicate a fundamental difference in the plasmaconcentration-time profile for both PEGylated photosensitisers, with SPC0172 showing lower absolute levels and more rapid elimination.

Other tissues were taken from the mice at the same timepoints as theblood samples and treated using a similar extraction and fluorescenceanalysis method to determine photosensitiser levels. Levels in livermapped the same trends as seen in plasma for the three photosensitisersFIG. 6 (nano-moles per g wet weight, w against time, t (hours)) showsliver photosensitiser concentrations following injection of 0.88nanomoles of each photosensitiser per g live weight. Once again liverlevels for SPC 0038B were higher and more persistent whereas levels forSPC 0172 were lower and eliminated more quickly.

Levels in skin are presented in FIG. 7 (nano-moles per g wet weight, wagainst time, t (hours)), skin photosensitiser concentrations followinginjection of 0.88 nanomoles of each photosensitiser per g live weight.Relatively high concentrations of m-THPC occurred in skin 24 hours afteradministration falling to lower levels from 72 hours onwards. SPC 0172concentrations in skin were low throughout the 6-192 hour measurementperiod suggesting a lower potential to cause cutaneous photosensitivity.Skin concentrations of SPC 0038B were higher than those of SPC 0172 orm-THPC between 72 and 192 hours suggesting a higher potential to causecutaneous photosensitivity during this period.

The selectivity for tumour specific tissue distribution was alsoevaluated in this work. Tissue selectivity was evaluated as thetumour:muscle concentration ratio over the duration of the observationperiod (4-192 hours post-administration). This ratio provided a directmeasure of the normal tissue to tumour tissue differential inconcentrations and allows selection of an optimal treatment time for PDTthat minimises the potential to cause collateral damage of normaltissues. The results obtained are presented in FIG. 8 (Tumour:muscleratio, r against time, t (hours)), Tumour:muscle concentration ratio ofthe three photosensitisers.

The results indicate an optimal tumour:muscle concentration ratio forSPC 0172, and hence tumour PDT, occurs at 72 hours post-administration.The ratio value of about 7.5 with SPC 0172 was similar to that thatobtained with SPC 0038B at the same timepoint and 2-3 fold higher thanthat observed with m-THPC. Both PEGylated photosensitisers thereforeexhibit improved tumour targeting in comparison to m-THPC. In comparisonto SPC 0172, the favourable tumour: muscle ratio for SPC 0038B does notdecline as rapidly beyond 72 hours suggesting a broader window fortumour PDT but also the less attractive feature of persistent PDTbioactivity in the target tissue. SPC 0172 exhibits the most appropriateprofile for tumour PDT of these two PEGylated photosensitisers with asingle optimum at 72 hours and low skin levels suggesting a reducedpotential to cause cutaneous photosensitivity.

Published work (Grahn et al, Proc. SPIE 1997, 3191, 180-6) with SPC0038B in this model support the observation that this photosensitiser iseliminated more slowly than m-THPC.

This work also showed that SPC 0038B concentrations measured in musclepeaked at or before 72 hours after injection whilst those in tumourpeaked between 72 and 144 hours, after which they declined. An optimaldrug-light treatment interval for tumour PDT specific tissue necrosislater than 72 hours post-administration was therefore indicated. Alliedmeasurements of tumour bioactivity showed sustained PDT tumour necrosisfrom SPC 0038B occurred for PDT treatment times ranging from 4-15 days.This report also confirms the persistence of liver concentrations of SPC0038B. These data further illustrate a phenomenon of persistent tumourlevels and sustained potential for PDT-mediated tumour necrosis with SPC0038B that are sub-optimal characteristics of an ideal PDT agent.

Cutaneous Photosensitivity

Cutaneous photosensitivity was determined in adult female Balb/c mice,held under standard conditions and allowed access to food and water adlibitum. Groups of mice of weight 19-22 g were given thephotosensitisers at a dose of 0.88 μmol kg⁻¹ by injection into the tailvein. The solutions for injection of each compound were prepared at aconcentration of 0.352 μmol ml⁻¹ so that a typical 20 g mouse would beinjected with a volume of 50 μl. Each mouse was weighed immediatelybefore injection and the injected volume adjusted to give the correctdose. For m-THPC the injection solution was prepared using the standardPEG400: ethanol:water vehicle, whilst SPC 0172 and SPC 0038B wereprepared in water for injection.

At 24 or 72 hours after photosensitiser injection one ear of each mousewas irradiated with full-spectrum xenon light from a 1 KW clinicalphoto-irradiator (Model UV-90, Applied Photophysics, London). The lightwas delivered to the ear using a 7 mm diameter light guide (Serial SU3)placed lightly against the ear. The light dose given was 40 Joules percm², which was achieved by exposing the ear for 63 seconds to 245 mW oflight from the light guide having a contact area of 0.384 cm². The lightguide filtered infra-red and ultra-violet light, so as to minimiseheating of the ear and damage resulting from ultra-violet lightirradiation.

Animals treated with light at the 24 or 72 hour drug-light intervalswere killed at 48 or 24 hours respectively after irradiation of theears. The oedema of the irradiated and control ears was assessed bymeasuring ear thickness. The thickness of the ear was measured at threesites on the upper third of the ear using a fixed-force micrometer witha vernier-interpolated resolution of 2 μm and an accuracy of 10 μm(Neill instruments).

The results indicate that ear swelling or thickness, as a surrogateendpoint for cutaneous photosensitivity, is lower with SPC 0172 thanwith SPC 0038B or m-THPC following light irradiation either at 24 or 72hours after photosensitiser administration (Table 2). SPC 0038Bexhibited superiority over m-THPC to cause less photosensitivity at the24 hour drug-light interval but this difference was negated evenreversed by the 72 hour drug-light interval. These observations matchthe trends in skin concentrations of these photosensitisers notedearlier. The indication therefore is that SPC 0172 is superior to SPC0038B or m-THPC in terms of potential to cause cutaneousphotosensitivity.

TABLE 2 Differences in ear thickness (irradiated-control) in μm for miceinjected with 0.88 μmol kg⁻¹ of each photosensitiser 24 or 72 hoursbefore light irradiation. 24 h drug-light interval 72 h drug-lightinterval Photosensitiser Mean SEM ^(1,2) N Mean SEM ^(1,2) N SPC 0038B25 7 (16 to 38) 3 4 6 (−2 to 15) 3 SPC 0172 2 (22; −18) 2 0 (8; −8) 2m-THPC 41 25 (8 to 90) 3 2 7 (−12 to 13) 3 ¹ Standard error of mean. ²Range or individual values provided in parentheses.Tumour PDT Activity

Information on the tumour PDT activity have been obtained in theaforementioned mouse model bearing the syngeneic colo26 tumour implantedsubcutaneously at the top of the left hind leg (approximately 1 cmlateral to the spine). The tumours were used after 12 to 16 days whenthey had reached an average diameter of 8 to 10 mm. For drug injectionand irradiation animals were sedated with Hypnorm diluted in water(1:3).

SPC 0172 demonstrated tumour necrosis at 1 day after drug injectionwhich increased in extent at two days post-injection (Table 3). Theeffect observed at two days post-injection comprised full tumournecrosis, an effect equivalent to that observed with m-THPC at half thedose in the same timescale. Examination of the treated sites with SPC0172 suggested that tumour necrosis was accompanied by only limiteddamage of the surrounding skin and underlying muscle, particularly atthe two day drug-light interval.

Further experiments using the same dose of SPC 0172 and a light dose of20 J cm⁻² were carried out at a range of drug-light intervals. Fullthickness tumour damage was observed at all drug-light intervals up to72 hours.

Comparative data on SPC 0038B at its optimal drug-light interval of 72hours in this model show 10-20 fold less potency on a dose level anddegree of tumour necrosis basis in comparison to SPC 0172 and m-THPC.

The results suggest that the in-vivo potency of SPC 0172 as a tumour PDTagent is similar to that of m-THPC at its optimal dose (0.88 μmol kg⁻¹)whereas SPC 0038B is somewhat less potent.

TABLE 3 Tumour necrosis induced in the mouse implanted colorectal tumourmodel by the 3 photosensitisers Light applied Biological Drug-light at652 nm (J effect Drug Dose interval cm⁻¹) at 100 (mm of tumour Compound(μmol kg⁻¹) (hours) mW cm⁻¹ necrosis) SPC 0172 1.76 24 20 3.8 ± 1.3 SPC0172 1.76 48 10 Full thickness (>6 mm) SPC 0038B 4.4 72 5 3.3 ± 0.7 ¹m-THPC 0.88 24 5 5.8 ± 0.4 m-THPC 0.88 48 5 5.6 ± 0.8 ¹ Taken from Grahnet al Proc. SPIE 1997, 3191, 180-6.

CONCLUSION

In summary, PEGylated photosensitisers exhibit improved water solubilitycompared to the parent photosensitiser m-THPC making them far morepharmaceutically acceptable for parenteral administration. They aretaken up by tumour cells in a different energy-independent mechanismcompared to m-THPC in which SPC 0172 is more efficient than SPC 0038B.Improved tumour targeting over m-THPC was achieved by these PEGylatedmolecules with SPC 0172 showing a superior pharmacokinetic profilecompared to SPC 0038B with a peak in tumour: normal tissue ratio within72 hours of administration and more rapid elimination from the tissuesand plasma. A lower potential to cause cutaneous photosensitivity wasevident with SPC 0172 over SPC 0038B or m-THPC and a similar potency tom-THPC for tumour necrosis following PDT by SPC 0172 was shown whereasSPC 0038B was less active. It is therefore apparent thatcarbamate-linked PEGylated photosensitisers as evidenced by SPC 0172 aremore ideal tumour PDT agents than triazine-linked PEGylatedphotosensitisers as evidenced by SPC 0038B, and possess improvedfeatures in comparison to m-THPC.

1. The compounds selected from: 7,8-Dihydro-5, 10,15,20-tetrakis(3-hydroxyphenyl)prophyrin derivatised with ω-methoxypolyethylene glycol (average MW=2000) via biscarbamate linkages derivedfrom hexane-1,6-diisocyanate; 7,8,17,18-Tetrahydro-5, 10,15,20-tetrakis(3-hydroxyphenyl)prophyrin derivatised with ω-methoxypoly(ethylene glycol) (average MW=2000) via bis carbamate linkagesderived from hexane-1,6-diisocyanate; 7,8-Dihydro-5, 10,15,20-tetrakis(3-hydroxyphenyl)prophyrin derivatised with ω-methoxypoly(ethylene glycol) (average MW=5000) via bis carbamate linkagesderived from hexane-1,6-diisocyanate; and 7,8,17,18-Tetrahydro-5, 10,15,20-tetrakis(3-hydroxyphenyl) prophyrin derivatised with ω-methoxypoly(ethylene glycol) (average MW=5000) via bis carbamate linkagesderived from hexane-1,6-diisocyanate.